Extension of human GCSF serum half-life by the fusion of albumin binding domain

Abstract

Granulocyte colony stimulating factor (GCSF) can decrease mortality of patients undergo chemotherapy through increasing neutrophil counts. Many strategies have been developed to improve its blood circulating time. Albumin binding domain (ABD) was genetically fused to N-terminal end of GCSF encoding sequence and expressed as cytoplasmic inclusion bodies within Escherichia coli. Biological activity of ABD-GCSF protein was assessed by proliferation assay on NFS-60 cells. Physicochemical properties were analyzed through size exclusion chromatography, circular dichroism, intrinsic fluorescence spectroscopy and dynamic light scattering. Pharmacodynamics and pharmacokinetic properties were also investigated in a neutropenic rat model. CD and IFS spectra revealed that ABD fusion to GCSF did not significantly affect the secondary and tertiary structures of the molecule. DLS and SEC results indicated the absence of aggregation formation. EC50 value of the ABD-GCSF in proliferation of NFS-60 cells was 75.76 pg/ml after 72 h in comparison with control GCSF molecules (Filgrastim: 73.1 pg/ml and PEG-Filgrastim: 44.6 pg/ml). Animal studies of ABD-GCSF represented improved serum half-life (9.3 ± 0.7 h) and consequently reduced renal clearance (16.1 ± 1.4 ml/h.kg) in comparison with Filgrastim (1.7 ± 0.1 h). Enhanced neutrophils count following administration of ABD-GCSF was comparable with Filgrastim and weaker than PEG-Filgrastim treated rats. In vitro and in vivo results suggested the ABD fusion as a potential approach for improving GCSF properties.

Investigation of the pH-dependent aggregation mechanisms of GCSF using low resolution protein characterization techniques and advanced molecular dynamics simulations

“…2 b). Surprisingly, GCSF at pH 7.5 shows a negative CD signal at wavelengths from 300 to 340 nm which is very unusual for a protein in inorganic buffer but has been previously observed for filgrastim [55] . It is presumably caused by aggregates in the sample.…”

Decoration of graphene oxide nanosheets with carboxymethylcellulose hydrogel, silk fibroin and magnetic nanoparticles for biomedical and hyperthermia applications

“…Finally, optical densities were measured at 550 nm using a microplate reader spectrophotometer (BioTeK, USA). 36 All tests were done in duplicate. EC50 values, the concentration of the substrate (here mg mL −1 ) at which 50% of maximum proliferation was achieved, were calculated using Prism software (v. 8.0).…”

“…1% SDS was added to the wells and incubated for 16 h at 37 °C. Finally, optical densities were measured at 550 nm using microplate reader spectrophotometer (BioTeK, USA) 36 . All tests were done in duplicate.…”

A Machine Learning Method with Filter-Based Feature Selection for Improved Prediction of Chronic Kidney Disease

“…It indicates the hydration status of a patient together with the functional ability of the patient’s kidney. Albumin is a protein found in the blood [ 36 ]. When the kidney is damaged, it allows albumin into the urine.…”